Cholera remains an important cause of illness in many developing countries and has been estimated to result in more than 200,000 deaths each year. Infection with enterotoxigenic E. coli (ETEC) is the most frequent cause of diarrhoea in the developing world and amongst travellers; it is responsible for more than one billion diarrhoeal episodes and one million deaths annually. Infection with ETEC is also an important cause of disease in animals For both cholera and ETEC infections there is a great need for effective vaccines.
In both cholera and ETEC infections, the primary cause of diarrhoea is the action of an enterotoxin released by the infecting organisms in the intestine; in the case of cholera cholera toxin (CT) and in the case of ETEC heat-labile enterotoxin (LT). The two toxins are closely related both structurally and functionally, each consisting of a toxic A subunit (CTA or LTA respectively) surrounded by five identical B subunits (CTB or LTB respectively) (Spangler, 1992). The B subunit pentamers are responsible for the binding of the toxin to GM1 ganglioside receptors present on the surface of intestinal epithelial cells (Holmgren, 1981); LT can also bind to structurally related galactoprotein receptors (Holmgren and Fredman, et al., 1982).
Although both proteins may exhibit internal variation in a few amino-acid residues, e.g. in human versus animal ETEC isolates and in classical versus El Tor biotype cholera strains, LTB and CTB show a high degree of homology with 85% conservation of amino acids in the mature protein (FIG. 1) and there is evidence from crystallographic studies that LTB and CTB pentamers are also structurally similar (Sixma and Pronk, et al., 1991, Sixma and Kalk, et al., 1993, Merritt and Sarfaty, et al., 1994). There is also a high degree of immunological cross-reactivity between the two molecules (Svennerholm and Wickstrom, et al., 1986) despite the fact that the majority of antibodies are directed against structural features of assembled pentamers; a further indication of the structural similarity between the two molecules.
CT has been found to be an effective oral immunogen that gives rise to intestinal IgA responses directed mainly against the B subunit. Furthermore, oral administration of CTB alone has also been found to effectively stimulate similar responses and especially in humans, CTB has been found to be a strong immunogen in the absence of either the adjuvant or toxic effects of the holotoxin. These responses are associated with high-level although relatively short-term (ca. 6-9 months) protection against challenge or natural infection with Vibrio cholerae and a much longer-lasting immunological memory (Svennerholm and Sack, et al., 1982, Svennerholm and Gothefors, et al., 1984, Svennerholm and Jertborn, et al., 1984, Clemens and Sack, et al., 1990, Quiding and Nordstrom, et al., 1991).
Antitoxin antibodies appear to act synergistically in their protective action together with intestinal IgA antibodies directed against bacterial cell-associated antigens such as the lipopolysaccharide (LPS) of V. cholerae O1 (Svennerholm and Holmgren, 1976) or of the novel serotype O139 (J. Holmgren, et al., unpublished). Based on these findings an oral vaccine against cholera has been developed consisting of CTB together with killed whole cells of V. cholerae O1 (Holmgren and Svennerholm, et al., 1992) which has given rise to protection lasting several years (Clemens and Sack, et al., 1990) and which is presently being modified to also include cells of the O139 serotype.
Large-scale field trials of the B subunit-O1 whole cell vaccine in Bangladesh demonstrated in addition to the long-term protection observed against cholera, significant short-term cross-protection against ETEC infection due to the CTB component (Clemens and Sack, et al., 1988(a)). Such protection against ETEC afforded by the cholera vaccine was subsequently confirmed in a study of Finnish tourists travelling to Morocco (Peltola and Siitonen, et al., 1991). Based on this a more broad-spectrum vaccine against ETEC has been developed in which CTB is used in conjunction with killed E. coli expressing the major colonisation factor antigens involved in adhesion to the intestinal epithelium (Svennerholm and .ANG.rhen, et al., 1991). The E. coli strains were included in order to provide immunity against ETEC strains releasing a heat-stable enterotoxin (STa) either alone or together with LT.
In areas where both cholera and ETEC are endemic it would be desirable to have a single vaccine that could effectively protect against both infections. This could in part be achieved by increasing the protection against ETEC afforded by the CTB component of the already licensed cholera vaccine. Although LTB and CTB show significant immunological cross-reactivity, neutralisation of LT by serum from CTB-immunised individuals is not as effective as the same serum is in neutralising CT (.ANG.hren and Wenneras, et al., 1993). Conversely, it is known that antisera against LT or LTB react in higher titre with LT than with CT (Svennerholm and Holmgren, et al., 1983). It is therefore possible that some neutralising epitopes in LTB are absent in CTB and vice versa. This is further indicated by the identification of LTB-specific neutralising antibodies (Svennerholm and Wickstrom, et al., 1986).
The inclusion of CTB in both the cholera and ETEC vaccines has led to the development of an expression system for the overproduction of recombinant protein that can be produced in large quantities and in the total absence of the toxic CTA subunit (Sanchez and Holmgren, 1989, Lebens and Johansson, et al., 1993). This has also opened the way to relatively simple procedures for the genetic modification of CTB such as the generation of protein fusions carrying foreign peptide antigens.
The present invention is based on modification by site-directed mutagenesis of the structural gene coding for CTB resulting in hybrid proteins in which LTB-specific epitopes were introduced into proteins that remained essentially CTB. thereby generating such hybrid molecules carrying LTB-specific epitopes in addition to cross-reactive and CTB-specific ones, in order to increase the immunological cross-reactivity. Such hybrid CTB/LTB molecules have been developed in order to provide a broad spectrum vaccine for the prevention or treatment of enterotoxigenic illness.